This proposal describes a preliminary study of adeno-associated virus (AAV) genome-releasing structural intermediates to determine capsid rearrangements that promote uncoating. We will optimize a viral DNA (vDNA) fluorescent dye-based, genome-release assay using heat-treatment as a surrogate model for cell factors that cue the trigger for uncoating. We will test a variety of heat-treatment conditions, to model environmental cues that AAVs encounter during infection such as glycan receptors, cations, pH changes and proteases in order to assess capsid priming for uncoating. We will use the following correlated methods: 1) cryo-electron microscopy (cryo-EM) to determine structures of icosahedral, genome-releasing capsid intermediate forms; 2) fluorescence- based vDNA dynamics assays to distinguish uncoated and DNase protected vDNA; and 3) mass spectrometry to detect changes in protease protection of capsid peptides. Specific Aim 1. Develop fluorescence-based assays to characterize AAV genome dynamics during vDNA uncoating. We will determine optimal heat-treatment (HT) conditions for AAV GR from intact capsid shells. To probe the influence of capsid diversity and receptor binding determinants on GR dynamics we will study wildtype rAAV2 and rAAV5, and receptor-binding-deficient capsid variants rAAV2-R585A/R588A and rAAV5-Y585V/L587T, for vDNA release following HT. Additionally, we will study vDNA release of heat- treated AAV in the presence of environmental cues AAVs encounter during infection. To assess the influence of these cues on the disassembly cascade, the following sample conditions and additives will be studied alongside heat-treatment: pH (4.5 - 7.5); divalent cations; and glycan receptors. Specific Aim 2. Characterize structure dynamics of DNA-releasing AAV capsids using cryo-EM. The primary goals of this aim are to obtain preliminary data for high resolution structure studies on heated AAV, and to determine the influence of receptor binding determinants on HT-induced structural dynamics. 3D EM structures will be determined for heat-treated rAAV2 (55C) and rAAV5 (75C), as well as two receptor binding deficient capsid variants, rAAV2-R585A/R588A and rAAV5-Y585V/L587T subjected to the same treatment conditions.